Vibrio cholerae causes the severe diarrheal disease cholera. We found that Hfq, an RNA-binding protein, is essential for Vibrio cholerae virulence. Deletion of hfq abolished V. cholerae colonization of the suckling mouse intestine, but had a minimal effect on growth in vitro and did not influence expression of known colonization factors. Thus, Hfq appears to control previously undescribed pathways essential for cholera pathogenesis. [unreadable] [unreadable] In E. coli, Hfq binds to numerous small untranslated RNAs (sRNAs), modulates their activities, and thereby controls expression of a wide variety of genes. Hfq also binds to some mRNAs in E. coli and alters gene expression directly. Although Hfq in V. cholerae probably acts by similar mechanisms, the distinct phenotypes of hfq V. cholerae and E. coli suggest that the proteins bind different sets of RNAs and control distinct regulons. No RNAs bound by V. cholerae Hfq and no V. cholerae sRNAs have been characterized to date. [unreadable] [unreadable] The goals of this R21 application are to identify pathways controlled by Hfq in V. cholerae, particularly Hfq-regulated genes that contribute to V. cholerae virulence, and to characterize the mechanisms controlling their expression. Experiments in Aim I - to define the Hfq regulon - will generate the first knowledge of Hfq-regulated effectors in V. cholerae. Experiments in Aim II - cloning of sRNAs and mRNAs that interact with Hfq - will utilize a new, unbiased approach for cloning interacting RNAs. Experiments in both Aims I and II will explore which of Hfq's interaction partners and downstream effectors contribute to V. cholerae virulence and thus illuminate currently unknown mediators of pathogenesis. Experiments in Aim III - to match sRNAs to the genes they regulate and characterize processes of Hfq dependent gene regulation - will create the foundation for detailed analyses of the mechanisms by which Hfq controls gene expression in V. cholerae. These studies will also facilitate disruption of Hfq-mediated pathways. As Hfq contributes to the virulence of several other Class B Priority Pathogens in addition to V. cholerae, Hfq or its downstream effectors may prove to be valuable targets for new antimicrobial agents. [unreadable] [unreadable]